Diverse ETS transcription factors mediate FGF signaling in the Ciona anterior neural plate - PubMed (original) (raw)

Diverse ETS transcription factors mediate FGF signaling in the Ciona anterior neural plate

T Blair Gainous et al. Dev Biol. 2015.

Abstract

The ascidian Ciona intestinalis is a marine invertebrate belonging to the sister group of the vertebrates, the tunicates. Its compact genome and simple, experimentally tractable embryos make Ciona well-suited for the study of cell-fate specification in chordates. Tunicate larvae possess a characteristic chordate body plan, and many developmental pathways are conserved between tunicates and vertebrates. Previous studies have shown that FGF signals are essential for neural induction and patterning at sequential steps of Ciona embryogenesis. Here we show that two different ETS family transcription factors, Ets1/2 and Elk1/3/4, have partially redundant activities in the anterior neural plate of gastrulating embryos. Whereas Ets1/2 promotes pigment cell formation in lateral lineages, both Ets1/2 and Elk1/3/4 are involved in the activation of Myt1L in medial lineages and the restriction of Six3/6 expression to the anterior-most regions of the neural tube. We also provide evidence that photoreceptor cells arise from posterior regions of the presumptive sensory vesicle, and do not depend on FGF signaling. Cells previously identified as photoreceptor progenitors instead form ependymal cells and neurons of the larval brain. Our results extend recent findings on FGF-dependent patterning of anterior-posterior compartments in the Ciona central nervous system.

Keywords: Ciona intestinalis; ETS; FGF; Neural patterning.

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Figures

Fig. 1

The Ciona mid-gastrula neural plate. (A) Schematic of a mid-gastrula stage embryo showing the organization of the 6-row neural plate. (B) Gene expression patterns and fates of neural plate territories. Rows V and VI express FoxC and give rise to the adhesive palps, oral siphon placode (OSP) and rostral trunk epidermal neurons (RTENs). Row IV expresses Six3/6 and contributes to the anterior sensory vesicle (ASV). The a9.49 cells in lateral row III express Tyrp1a and give rise to pigmented cells (PC). Medial row III cells (a9.33 and a9.37) express Myt1L and also contribute to the ASV. The _FGF8_-expressing A9.30 cells in lateral row II form the motor ganglion (MG), whereas medial row II forms posterior sensory vesicle (PSV). Row I contributes to the tail nerve cord. (C, D) Co-electroporation of Dmrt and ZicL reporters allows for tracing of cells from the mid-gastrula neural plate (C) to the late tailbud stage (D). Colored lines in (D) indicate territories derived from rows labeled with the corresponding color in (C). Rows V and VI express Dmrt exclusively and contribute to the palps, OSP, and peripheral nervous system. ZicL and Dmrt reporters co-localize in rows III and IV, which give rise to the anterior sensory vesicle. Rows I and II express ZicL exclusively and contribute to posterior brain, motor ganglion, and tail nerve cord. Scale bars in (C, D) = 50 µm.

Fig. 2

MEK/FGF-dependent patterning of rows III and IV of the anterior neural plate. (A–D) Embryos were electroporated with Dmrt > LacZ and treated with DMSO (control) or MEK inhibitor U0126 at the 110-cell stage, then fixed at mid-gastrula stage and processed for in situ hybridization with probes against Six3/6, MyT1L, and Tyrp1a and immunostaining with anti-β-galactosidase antibody (αβ-gal). In control embryos, Six3/6 expression is restricted to row IV (A). Myt1L is expressed in medial row III and Tyrp1a is expressed in the lateral a9.49 cells of row III (B). After treatment with U0126, Six3/6 expression expands posteriorly into row III (C) at the expense of Myt1L and Tyrp1a (D). (E–H′) Embryos electroporated with ZicL > YFP-CaaX alone or co-electroporated with ZicL > dnFGFR, fixed at mid-gastrula stage, and processed as in (A–D) except using anti-GFP antibody (αGFP). In control embryos, wild-type patterns of Six3/6, Myt1L, and Tyrp1a expression are observed (E–F′). ZicL > dnFGFR results in posterior expansion of Six3/6 and loss of Myt1L and Tyrp1a, recapitulating U0126 treatment (G–H′). (E–H) ISH overlayed with immunostaining and DIC channels. (E′–H′) ISH channels alone. Embryos in (A, C, E, G, H) are left–right mosaics, owing to unequal inheritance of electroporated plasmids; embryo in (B) also exhibits mosaic inheritance of the Dmrt reporter. Scale bars = 50 µm. Asterisks indicate expression of Myt1L in row I. See full quantitation of observed phenotypes in Supplementary Fig. 5.

Fig. 3

Partially redundant roles for Ets1/2 and Elk1/3/4 in the anterior neural plate. (A–D′) Embryos were co-electroporated with ZicL > YFP-CaaX and ZicL > Ets:WRPW (A–B′) or ZicL > Ets:VP16 (C–D′), and then assayed for expression of Six3/6 (A, A′, C, C′), or Myt1L (B, B′, D, D′) at mid-gastrula stage by in situ hybridization and immunohistochemistry with GFP antibody (αGFP). ZicL > Ets:WRPW leads to modest expansion of Six3/6 (A, A′) and loss of Myt1L (B, B′). ZicL > Ets:VP16 embryos show mostly wild-type expression of Six3/6 (C, C′) and a moderate expansion of Myt1L (arrows in D, D′). (E–H′) Similar to (A–D′), except embryos were electroporated with ZicL > Elk:WRPW (E–F′) or ZicL > Elk:VP64 (G–H′). ZicL > Elk:WRPW has a slightly stronger effect on Six3/6 expansion into row III (E, E′). This expansion occurs at the expense of Myt1L (F, F′). ZicL > Elk:VP64 results in repression of Six3/6 in row IV (G, G′) and expansion of Myt1L expression (arrows in H, H′). Embryos in (B, B′), (F, F′) and (G, G′) are left–right mosaics, owing to unequal inheritance of electroporated plasmids. Scale bar = 50 µm. Asterisks indicate expression of Myt1L in row I. See full quantitation of observed phenotypes in Supplementary Fig. 5.

Fig. 4

Ets1/2 and Elk1/3/4 have distinct larval phenotypes. (A–B) Control and FGF-inhibited larvae. (A) Control larvae possess two pigmented cells within the sensory vesicle. (B) ZicL > dnFGFR results in complete loss of pigmented cells in the majority of larvae. (C–D) Misexpression of Elk1/3/4 fusion proteins. (C) ZicL > Elk:VP64 larvae contain 2 pigmented cells, comparable to controls. (D) ZicL > Elk:WRPW results in fewer pigmented cells relative to controls, similar to ZicL > dnFGFR. (E, F) Misexpression of Ets fusion proteins. (E) ZicL > Ets:VP16 is sufficient to produce supernumerary pigmented cells, whereas ZicL > Ets:WRPW results in loss of pigmentation (F), as previously described. (G, H) Larvae electroporated with Tyrp1a > LacZ (G) or Tyrp1a > Six3/6 (H). Misexpression of Six3/6 in the pigmented cell lineage is sufficient to abolish pigmentation. Scale bars = 50 µm.

Fig. 5

A revised origin of Ciona photoreceptor cells. (A, B) Control and ZicL > dnFGFR embryos were co-electroporated with reporters for Six3/6 and Arrestin, and then grown to larval stage. (A) Arrestin reporter labels a cluster of cells surrounding the ocellus, posterior to the Six3/6 expression domain. (B) Inhibition of FGF signaling results in posterior expansion of the Six3/6 reporter, but no significant change in Arrestin reporter expression. Arrestin reporter does not colocalize with ectopic Six3/6 reporter. (C, C′) Embryos were co-electroporated with reporters for Dmrt and Arrestin and grown to larval stage. Clusters of _Arrestin_+ cells are consistently observed posterior to the Dmrt expression domain. Scale bars = 50 µm.

Fig. 6

Summary of FGF-dependent patterning in the mid-gastrula neural plate. FGF signaling at the 112-cell stages leads to asymmetric division of the row III/row IV precursors. Activated Ets1/2 and Elk1/3/4 lead indirectly to repression of Six3/6 in row III and expression of Myt1L in row I and medial row III. These _Myt1L_+ cells contribute to ependymal cells of the tail nerve cord (TNC) and sensory vesicle (SV). Ets/Pnt2 mediates expression of Tyrp1a and ultimately promotes pigment cell formation in the lateral a9.49 lineage of row III.

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Diverse ETS transcription factors mediate FGF signaling in the Ciona anterior neural plate - PubMed (original) (raw)